Autores: Luzia Cristina Lencioni Sampaio, Thirssa Helena Grando, Lucas Trevisan Gressler, Matheus Dellaméa Baldissera, Dianni de Menezes Capeleto, Mariângela Facco de Sá, Francielli Pantella Kuns de Jesus, Alceu Gonçalves dos Santos Junior, Andreia Nobre Anciuti, Karina Colonetti, Daniel Roulim Stainki, Silvia Gonzalez Monteiro*.
De acordo com normas para publicação em: Veterinary Parasitology
Artigo submetido para publicação na Revista “Veterinary Parasitology” (Anexo 2)
Production, purification and therapeutic potential of egg yolk antibodies for treating Trypanosoma evansi infection
Luzia Cristina Lencioni Sampaioa*, Thirssa Helena Grandoa, Lucas Trevisan Gresslera, Matheus Dellaméa Baldisseraa, Dianni de Menezes Capeletoa, Mariângela Facco de Saa, Francielli Pantella Kuns de Jesusa, Alceu Gonçalves dos Santos Juniorb, Andreia Nobre Anciutib, Karina Colonettib, Daniel Roulim Stainkia, Silvia Gonzalez Monteiroa*.
a Department of Microbiology and Parasitology, Universidade Federal de Santa Maria (UFSM), Brazil.
b Biotechnology Center, Universidade Federal de Pelotas (UFPel), Brazil.
*Corresponding author at: Departamento de Microbiologia e Parasitologia - UFSM. Faixa de Camobi - Km 9, Campus Universitário, Santa Maria - RS, Brasil. 97105-900, Prédio 20, Sala 4232
Tel: +55 55 3220-8958
Abstract
The use of avian antibodies has aroused interest in biomedical research due to the numerous advantages compared to mammal’s antibodies. Our study aimed to produce and purify IgY immunoglobulins in order to use as an alternative therapy against Trypanosoma evansi. Every 14 days, four New Hampshire chickens were immunized with trypomastigotes of T. evansi, totaling five inoculations. Eggs were collected during 70 days and the extraction of IgY was performed by precipitation through the PEG- 6000 method. Characterization and purification of IgY anti-T. evansi was carried out by SDS-PAGE and Western blot, where heavy and light chains were detected. The dosage of total proteins determined by photometry at 280 nm, obtaining an average yield production of 9,46 mg/mL. Sample´s titration allowed the quantification of specific IgY anti-T.evansi, with antibodies produced showing high avidity indexes. The results indicated that T. evansi is able to generate an immune response in poultry, resulting in a production of specific antibodies. In the in vivo study, IgY treatment resulted in increase of pre-patent period and in the longevity, when compared with the positive control, demonstrating an initial, but no curative, trypanocidal activity.
Keywords: immunotherapy, avian immunoglobulin, IgY, tripanosomosis.
Introduction
Trypanosoma evansi (T. evansi) is the etiologic agent of the disease known in Brazil as trypanosomosis, “Mal das Cadeiras” or “Surra”, which affects horses of all breeds and has worldwide distribution (Darling, 1910; Hoare, 1972). Cattle, sheep, goats, donkeys, cats and pigs are also susceptible hosts. Canines, capybaras (Hydrochaeris hydrochaeris), coatis (Nasua nasua) and vampire bats (Desmodus rotundus) are reservoirs and occasionally may manifest clinical symptoms; additionally, bats are considered vectors of this disease (Nunes et al., 1993; Silva et al., 2002). The transmission of the parasite is essentially mechanical, since trypomastigotes are transferred from one host to another through blood-sucking insects (flies of the families Tabanidae and Muscidae) or artificially by needles contaminated with infected blood (Silva et al., 2002). Clinical signs include weight loss, pale mucous membranes, intermittent fever, cough, swelling in the lower parts of the body, superficial lymph nodes increased, muscle atrophy, incoordination, hindquart’s paresis, difficulty to get up and proprioceptive deficits (Levine, 1973; Aquino et al., 1999; Herrera et al.; 2005). Chronic infections can last for years (Brun et al., 1998), usually occurring at this stage the worsening of the clinical signs and cachexia (Brandão et al., 2002; Silva et al., 2002; Rodrigues et al., 2005).
In Brazil, the most commonly drug used in trypanosomosis treatment in domestic animals is the diminazene aceturate, which is able to provide an elimination of the trypanosomes in bloodstream, just few hours after its administration (Peregrine and Mamman, 1993). However, it do not presents curative efficacy, sometimes occurring parasitemia relapses, mainly because most of the trypanocidal drugs do not cross the blood brain barrier (Lonsdale-Eccles and Grab, 2002; Masocha et al., 2007).
The production of polyclonal antibodies, by poultry immunization, has aroused interest in scientific community. In addition to the numerous advantages described in literature (Olovsson and Larsson, 1993; Svendsen et al., 1996; Contreras et al., 2005; Schade et al., 2005) for antibodies produced in mammals, the IgY technology is an ethical experimental procedure by replacing the bleeding for collecting eggs. The process consists in the immunization of chickens at regular intervals, with subsequent production of serum immunoglobulin (IgG). These proteins are transferred to the yolk and are named IgY (Y=yolk). The amount of IgG in the yolk is dependent upon its
concentration in serum (Carlander et al., 2002; Tini et al., 2002). The time between immunization and detection of antibodies in yolks is variable, depending on the biological assay performed (Patterson et al., 1962; Schade et al., 2005). The extraction and purification of it from yolk involves two mechanisms: delipidation and aqueous extract purification (Staak et al., 2001).
The present study aims to produce highly effective and pure antibodies by immunizing chickens with trypomastigotes of T. evansi; evaluate the therapeutic efficacy of these antibodies in rats experimentally infected with this protozoan; compare the therapeutical responses among rats treated only with IgY-anti T. evansi and rats treated with this antibody in association with diminazene aceturate and imidocarb dipropionate
Material and Methods
All procedures in this study were approved by the Animal Welfare Committee of Ethics in Animal Experimentation of Universidade Federal de Santa Maria (UFSM), number 018/2013.
2.1 IgY production and characterization
The antigen
For this experiment, it was used a strain of T. evansi obtained from a dog naturally infected (Colpo et al., 2005), which is kept in liquid nitrogen. Prior of each immunization, the parasites were thawed and injected into rats, intraperitoneally (Silva et al., 2003). When the rats presented high parasitemia, their blood was collected and the parasites were counted using a Neubauer chamber (Wolkmer et al., 2007).
Chicken immunization
Four 30-days-old egg-laying New Hampshire hens were kept in individual cages, receiving food and water ad libitum until the beginning of the egg laying. For
inoculation, a solution containing at least 107 tripomastigotes of T. evansi was used, added to incomplete Freund’s adjuvant (Sigma-Aldrich®) at a 1:1 ratio, with a final volume of 1.0 mL. Inoculations were performed in three chickens, intramuscularly, at five different sites of the pectoral muscle (200 μL/site). The first immunization was performed so that the hens began posture. The interval between immunizations was set as 14 days (days 0, 14, 28, 42 and 56), totaling five inoculations. The fourth hen was not immunized, serving as the negative control.
Selection and initial processing of eggs
From the fourth week post-immunization (day 21), the eggs were collected daily until the 70th day. They were all identified and stored at 4 ºC for further purification and antibodies characterization.
Extraction of immunoglobulin IgY anti-T. evansi
Extraction was performed by the method of precipitation of polyethylene glycol 6000 (PEG-6000) as described by Polson et al. (1980) and Pauly et al. (2011). After separation of white and yolk, the egg yolk was transferred to a filter paper and after to a 250 mL tube. The delipidation was performed through the emulsion of the yolk in PBS at 2:1 proportion. After this step PEG-6000 at 3,5 % was added. The mixture was then centrifuged at 4 ºC, 13,000 x g for 20 minutes. The supernatant obtained was transferred to a new tube, while the lipid layer was discarded. The process was repeated twice using PEG-6000 at 8,5 % and 12 %, respectively. After dialysis, the obtained extract was transferred to a 2 mL eppendorf tubes and stored at -20 ºC.
Total protein concentration
The determination of protein content was performed by photometry at 280 nm, with the samples diluted in PBS (1:50). To estimate the amount of IgY anti-T. evansi produced a calculation according to the Lambert-Beer law was applied, using an extinction coefficient of 1,33 to IgY (Pauly et al., 2011).
SDS-PAGE - Polyacrylamide gel electrophoresis
Purified IgY was electrophoresed on SDS-PAGE using 10 % polyacrylamide gel to check the purity, according to Laemmli (1970). The extracted samples were applied to the stacking gel wells in a final volume of 10 μL. The electrophoresis, subjected to an initial current of 80V and 120V, was stopped when the dye used in the samples reached the base of the separating gel. One removed from the system, the gel was stained with Comassie blue R 250 (Sigma-Aldrich®) for at least 1 hour and, then, treated with a decolorizing solution (glacial acetic acid 100 mL + methanol 400 mL + distilled water 500 mL) for visualization of protein bands (Bernardo, 2009). In another assay, the sample with trypomastigotes forms of T. evansi (antigen) were subjected to SDS-PAGE under the same conditions described above.
Western blot
The IgY extracted was subjected to polyacrylamide gel of electrophoresis as described above, being electro transferred to a nitrocellulose membrane 0,45 µm (Bio-Rad®). For the transfer it was used electroblotting mini tank (30V), which remained in cold chambering “overnight”. The membrane was blocked with 5 % skim milk in PBS-T for 1 hour. The membrane was washed 3 times with PBS-T and incubated for 1 hour with rabbit anti-chicken IgY peroxidase conjugate (1:2000) (Sigma-Aldrich®, USA). Finally, the membrane was washed with PBS-T 3 more times. The reaction was revealed with diaminobenzidine prepared in Tris HCl + nickel sulphate + hydrogen peroxide. The membrane remained under constant stirring until visualization of the reactive bands. In the assay, in which antigen was subjected to electrophoresis in polyacrylamide gel, electro transfer to nitrocellulose membrane was identical as described above. After blocking the membrane, it was washed 3 times with PBS-T and incubated for 1 hour with IgY extracted from the samples (1:50). The membrane was washed 3 times again and incubated with rabbit anti-chicken IgY peroxidase conjugate (1:2000) for more 1 hour. All incubations were carried out at room temperature. The reaction was revealed as described previously.
Double Radial Immunodiffusion in agarose gel
In order to perform the test, initially it was prepared a borate (boric acid 0,09 g + potassium chloride 0,36 g + water 50 mL), and the agarosis gel (agarose Sigma 0,4 g + borate buffer 2,5 mL + saline 0,85 % 42,5 mL). In a Petri dish of 85 mm diameter 15 mL of agarose gel was placed. After this, was prepared a test pattern of seven wells for drilling the gel to the bottom of the plate (a central cavity around which six wells arranged in a circle). In the central cavity antigen was placed. The peripheral wells 1, 3 and 5 were filled with negative control (Milli-Q water) and the cavities 2, 4 and 6 with the extracted sample (IgY). The cavities were filled until the disappearance of the meniscus. The system was kept in BOD incubator, with reading at 24, 48 and 72 hours. The formation of a precipitation line shows the antigen recognition by the antibody.
ELISA (Enzyme Linked Immune Sorbent Assay)
96-well ELISA plates (Corning Costar Corporation, Cambridge, MA - USA) were sensitized with soluble antigen (10 µg/well) diluted in 0.05 M carbonate buffer, pH 9.6, and incubated "overnight" at 4 ºC. After incubation, the plates were washed 4x with saline buffer solution (SBS) (pH 7.2). For testing, the plates were first blocked with 100 µL per well, Fetal Bovine Serum - FBS - (Cripion®, SP, Brazil) (FBS + SBS) and incubated at 37 °C. After this incubation, the plates were washed again 4x with SBST (SBS pH 7.4 with 0.05 % of Tween 20). The test sera were diluted at 1:10, 1:100, 1:500, 1:2000, 1:3000, 1:5000 in FBS + SBS, distributed on the plates (100 µL/well), and incubated for one hour at 37 ºC. Followed this last incubation, the plates were washed 4x. Each test sample was subjected to three replicates per plate and 2 inter- plate repetitions. After incubation with the primary antibody the plates were washed 4x with SBST and subjected to incubation with a secondary antibody (IgY anti-peroxidase conjugate) (Sigma Aldrich®, USA) at 1:3000 dilution and incubated for one hour at 37 °C. Finally, the plates were washed again to, then, receive 100 mL of chromogenic substrate (orthophenylene-diamine, OPD). After fifteen minutes, the reaction was blocked with 10 µL of H2SO4 (4N), with reading held in microplate spectrophotometer with 490 nm filter.
ELISA avidity
To perform the test, 96-well ELISA plates (Corning Costar Corporation, Cambridge, MA - USA) were sensitized with antigen and incubated with IgY (1:3000), as described in the previous section. Then, in each well it was added 100 μL of 6M urea in SST, pH 7.2 for five minutes. Plates were washed three times with SSTT and the secondary antibody added. Revelation was performed as described above.
The results were expressed as the avidity index (AI)determined by the ratio between optical density values of samples treated with urea (U+) and the optical density of untreated samples (U-), and expressed in percentage (AI = U+/U- x 100). The values (AI) < 40 % were considerate of low avidity, AI between 41 and 70 % of medium avidity and AI > 70 % of high avidity.
Specific IgY concentration
Specific IgY anti-T.evansi content was measured by the Coomassie Blue method according to Bradford (1976), using bovine serum albumin as the standard. This assay is based on the binding of the dye Coomassie Blue G-250 to the protein. This binding is accompanied by measuring the absorbance maximum of the solution at 595 nm.
In vivo test
Experimental animals
Sixty rats (Rattus norvegicus), male, 90 days old and weighing an average of 346 (± 27.53) grams were used as the experimental model. They were kept in cages, six rats each, housed on a light/dark cycle of 12 h, and in an experimental room with temperature and humidity controlled (25 ºC; 70 % respectively). They were fed with commercial ration and water ad libitum. All animals were submitted to a period of 10 days for adaptation.
Animal’s infection
During the first step one rat (R1) were infected intraperitoneally with infected blood, kept cryopreserved in liquid nitrogen. This procedure was performed to obtain a large amount of viable parasite for infection of experimental groups. Animals in the groups A, C, D, E, F, G, H, I and J were inoculated intraperitoneally with 0.1 mL of blood from R1 containing 104 trypanosomes (Day 0). The animals were daily monitored through peripheral blood smear (Silva et al., 2006), and when it was observed an average of eight protozoan/field (microscopically), the treatment was started
Experimental design
Immunoglobulin IgY anti-T. evansi was administered intraperitoneally (IP), while imidocarb dipropionate and diaceturate of diminazene were administered intramuscularly (IM).
The rats were divided into a ten groups (groups A to J) with six animals each group. They were named and treated as follows: Grup A (positive control - PC): infected and not-treated animals; Grup B (negative control - NC): uninfected and not-treated animals; Grup C (IgY): infected and treated (IgY anti-T. evansi at 10 mg.kg-1), during five days; Grup D (IgY-Imid): infected and treated animals (imidocarb dipropionate 12 % [Imizol®1] at 3 mg.kg-1; plus IgY anti-T. evansi at 10 mg.kg-1), also during Five days; Grup E (IgY-Dim): infected and treated animals (diminazene diaceturate 7 % [Ganaseg®]2 at 3,5 mg.kg-1; and specific IgY anti-T. evansi at 10 mg.kg-1), during five days; Grup F (Imid): infected and treated animals (imidocarb dipropionate 12 % at 3 mg.kg-1), during five days; Grup G (Dim): infected and treated animals (diminazene diaceturate 7 % at 3,5 mg.kg-1), during five days; Grup H (Prev) or prevention: treated with specific IgY at 10 mg.kg-1 during the five days prior to the infection; Grup I (Tox) or toxicity: uninfected and treated with specific IgY anti-T. evansi (10 mg.kg-1) for five days; Grup J (30 mg): animals treated with IgY anti-T. evansi (30 mg.kg-1) , during five days (prior infection) and ten days (after de infection).
The animals were daily monitored during 90 days, following the technique described by Silva et al. (2006).
Data analysis
Data from experiments were analyzed by the Student t test and by one-way ANOVA, followed by the Tukey test when F was significant (P < 0.05)
Results
Total protein concentration
Considering the date obtained between the 4th and 6th and 7th and 10th weeks post- immunization (WPI), among all the chickens, significant difference was observed in protein production after the 7th WPI (Figure 1). Between the 4th and 6th WPI the average yield observed was 5.56 ± 1.5 mg/mL over the 7th and 10th weeks, where the average yield observed was 12.26 ± 2.37 mg/mL [t (3) = - 4.56; P<0.05]. Throughout the experimental period, the average concentration of total protein was 9.46 ± 1.58 mg/mL. It was observed a significant increase in protein production between the 6th and 7th weeks [t (3) = - 5.61; P<0.05]. From the 7th week on, the values remained increased until the end of the experiment (10th week).
When the chickens were evaluated separately, at the 4th to 6th weeks, the hens 1, 2 and 3 yielded an average of 7.16 mg/mL ± 2.28, 3.84 ± 1,02 mg/mL and 6.14 ± 0,82 mg/mL respectively, while from the 7th week their respective average was 10.59 ± 5.03 mg/mL, 11.23 ± 2.09 mg/mL and 14.96 ± 5.66 mg/mL. When evaluating protein production individually, no significant differences were observed among the three hens between the 4th and 6th weeks, as well as between the 7th and 10th weeks (P>0.05).
Gel electrophoresis SDS-PAGE e Western blot
The characterization of IgY was performed by SDS-PAGE and Western blot. By electrophoresis in 10% polyacrylamide, under reducing conditions, it was possible to observe that the three immunized chickens produced a well-stained peptide band, with molecular weight ranging between 75-50 KDa (Figure 2a). The recognition of peptide bands was performed by Western blot, where the IgY antibodies anti-T.evansi reacted with secondary antibody conjugated with peroxidase, showing a strong and specific
peptide band, with molecular weight ranging between 75-50 KDa, (heavy chain of IgY) and 25-20 KDa (light chain of IgY) (Figure 2b). The assay in that the antigen was electro transferred to a nitrocellulose membrane, the antigen-antibody reaction was characterized after addition of a secondary antibody. Due to the high concentration of antigen in sample, the reaction was identified immediately bellow the line of the stacking gel.
Double Radial Immunodiffusion in agarose gel
The samples taken from all immunized chickens responded positively to the presence of the antigen, forming well defined precipitation bands (figure 3). In the wells, where were deposited negative controls (non-immunized chicken sample), no reactivity with the antigen was observed.
ELISA
At all tested dilutions, positive results and producing IgY were observed throughout the study period (figure 4). Significant differences were observed in O.D. at 1:10 dilution between the 4th (1.04 ± 0.26) and 10th weeks (1.56 ± 0.12) [t (3) = -5.72; P<0.05]. There was also a significant difference between the 4th (1.20 ± 0.31) and 10th week (1.39 ± 0.22) [t (3) = - 5.04; P<0.05]. at a dilution of 1:100. In other tested dilutions, no significances were observed (P>0.05).
ELISA avidity
This test aims to determine the strength of binding of the antibody to the antigen at both sites. The analysis of avidity index revealed a significant difference between the antibodies produced on the 4th week (52.53 ± 13.2) compared to those produced on the 10th week (72.58 ± 5.1) at 1:100 dilution [t (3) = - 4.77; P<0.05]. It was also observed that increasing avidity occurred as the immunizations were performed (figure 5). All chickens showed increased rates of avidity, without statistical difference between
them, in the last week of the experiment. The other dilution showed no significant difference between the 4th and 10th weeks (P>0.05).
IgY anti- T.evansi concentration
It was noted the production of IgY during the whole period (figure 6). There was significant difference between 4th and 10th week at 1:500 dilution. The average production for the period was 2.64± 0.15 (4th week) and 2.87 ± 0.14 (10th week) [t (3) = -4.31; P<0.05].
In vivo tests
Table 1 shows the results regarding to the pre-patent period, longevity and time of deaths. Among all the T. evansi infected groups, it was found statistically significant differences in the pre-patent period for the group J, when compared with all the other groups. While the average detection of the parasite in the bloodstream was 1 to 1.6 days PI, in group J the parasitemia was detected between the 12th and 17th days (P <0.05)
The longevity analysis of all the infected and treated groups showed a statistically significant difference, compared with the positive control group, exception observed for the H group (prevention). The C, D, F and J groups did not control the infection, but significantly increased the longevity of their animals. The H group did not control the infection, showing no increase in its longevity. Only in the groups E and G the parasitemia was fully controlled during the monitoring period, did not occuring recurrence of infection throughout the observation period. Comparison of longevity among rats treated with imidocarb and rats treated with the combination imidocarb + specific IgY, also resulted in a statistically significant difference (P <0.05). The same result was not found between groups E and G, respectively treated with the association of antibody with diminazene aceturate and chemical drug alone (P> 0.05). The experimental protocols used in groups C and J resulted in a significant statistical difference between them, which was evidenced by the increase in the pre-patent period and longevity.
Discussion
The experimental production of antibodies depends on a correct immunization protocol. Ferella et al. (2012) tested two immunization protocols in poultry using variable dose of the antigen and the number of immunizations, concluding that the amount and timing of detecting the yolk IgY is dose-dependent. In our study we used the antigen at concentrations above 2 x 107 associated with the FIA, every 14 days. In